Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development. Author summary The biosafety level-3 (BSL-3) classification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impedes the research and antivirals development. We report a novel cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP) that can be used at BSL-2 laboratory for high-throughput neutralization and antiviral screening. This system consists of two components: a genomic viral RNA containing a deletion of nucleocapsid (N) gene, and a producer cell line expressing the N protein. The complete viral life cycle can be achieved and exclusively confined in the producer cells. Moreover, intein-mediated protein trans-splicing that splits N into two vectors further secures the biosafety of this system. Based on this system, we found residue-specific phosphorylation of N protein is critical for viral infection. Besides, high-throughput antiviral screening was developed and new drugs were discovered. Thus, this experimental system will facilitate the studies of SARS-CoV-2 biology and its antiviral development.
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